Phenazines are pigmented secondary metabolites produced by Pseudomonas aeruginosa. Long believed to be innocuous, phenazines are now known to have significant and deleterious biological effects on host organisms. Phenazines enhance the ability of P. aeruginosa to compete and aid the bacteria in outwitting host defenses in ways that contribute to the notoriously persistent infections caused by this organism. The long term objective of this research is to evaluate the feasibility of disabling the phenazine biosynthetic pathway and assessing whether this could be an effective strategy for controlling P. aeruginosa, most likely in combination with traditional antibiotic therapy. The overall goals of this research are to determine the chemical mechanisms of phenazine antibiotic biosynthesis through the use of structural and mechanistic studies that will fill gaps in our understanding and permit evaluation of the feasibility of developing specific inhibitors of phenazine biosynthesis. The initial tricyclic phenazine backbone is synthesized form chorismic acid by seven core enzymes, three of which (PhzD, -F, and -G) we recently examined in crystallographic and mechanistic studies. The specific aims of this project are to evaluate the enzymology and structural biochemistry of three additional enzymes. We will examine PhzE, which catalyzes the first committed step in phenazine biosynthesis, and two key phenazine modifying enzymes, PhzM and PhzS, that produce pyocyanin, a phenazine with significant bioactivity. Using X-ray crystallography, we will determine the three- dimensional structures of these proteins. Enzymology studies will focus on the substrate specificity and mechanistic properties of each enzyme that contributes to their ability to efficiently produce phenazine pathway intermediates. The results will provide a framework for future studies that will address the design of phenazine biosynthesis inhibitors and the feasibility of using them as therapeutics. The opportunistic pathogen Pseudomonas aeruginosa is a serious threat to individuals whose immunity is compromised by cystic fibrosis, burns, cancer, AIDS, and other conditions. P. aeruginosa accounts for 10 percent of nosocomial infections in the United States and mortality rates range from 20 percent to as high as 70 percent for those with the most serious underlying health problems. New strategies that complement existing antimicrobial therapies clearly need to be developed to more effectively combat P. aeruginosa infections.